硫化氢对精子发生及功能障碍的保护作用及机制的研究

发布时间：2018-04-28 05:10

本文选题：硫化氢 + 精子　；参考：《南京医科大学》2014年博士论文

【摘要】：男性不育是造成人类生殖障碍的重要原因。不育男性中超过85%均能够产生精子,但是大多质量低下。精子发生过程异常,会影响精子的功能,造成男性不育。除了基因突变和染色体异常等遗传缺陷,环境因素和感染等疾病因素是导致精子发生和功能障碍的主要原因。这其中氧化应激与炎症反应是最关键的损伤机制。目前为止,男性不育在临床上缺乏有效的或者针对性的治疗手段。硫化氢(Hydrogen Sulfide, H2S)是继一氧化氮(Nitic Oxide, NO)和一氧化碳(Carbon Monoxide, CO)后的第三种气体信号分子,在哺乳动物体内,H2S主要由胱硫醚-γ-裂解酶(CSE)、胱硫醚-pˉ合成酶(CBS)催化L-半胱氨酸生成。研究证实,生理浓度的H2S对于清除氧自由基,控制机体的氧化应激水平,对抗炎症反应具有十分重要的生理意义。在一些病理状况下,外源性补充H2S,可以明显减轻氧化应激和炎症造成的组织和器官的功能障碍。在关于女性生殖的研究中,H2S对于卵细胞的发育和成熟具有重要意义,并对胎儿的出生和孕妇心血管功能的保护具有重要价值。在男性的生殖系统中,同样也存在着内源性的H2S合成酶,但对于其生理意义的研究报道多局限于改善男性的勃起功能障碍。内源性的H2S合成酶在男性的睾丸和精子中均有表达,可能对于调控男性精子发生和精子功能具有重要的潜在价值。目前为止,H2S对男性生育障碍的治疗和预防,以及对人类精子质量的影响尚无报道。本研究的主要目标是探讨H2S对精子发生过程及精子功能是否具有保护作用,并进一步研究其发挥作用的可能机制。我们首先使用自由基检测仪评价了H2S在正常人精浆和育力低下病人精浆中含量的差异,用Western-blot的方法比较了内源性H2S合成酶在不同来源精子中表达的差异。结果表明低水平H2S的精浆常常伴随着较差活力的精子,弱精子中内源性H2S合成酶CBS的表达显著降低,提示H2S可能参与了男性不育的病理过程。我们用动物实验进一步验证H2S对男性生殖的意义：我们利用LPS诱导的炎症和STZ诱导的糖尿病,建立常见的精子发生障碍模型,建模成功后,腹腔注射外源性给予GYY4137或NaHS两种不同的H2S供体,给药结束后,用电脑辅助精液分析仪(CASA)评价小鼠附睾尾部的精子质量,并用HE染色观察小鼠睾丸生精结构的变化,结果表明H2S可以显著抑制炎症和糖尿病引起的精子质量的下降并改善睾丸结构的损伤。我们用Real-Time PCR和Western-blot的方法检测了内源性H2S合成酶CSE和CBS的mRNA水平和蛋白表达水平,并用化学法检测了睾丸中CBS和CSE的催化活性,结果显示外源性给予H2S可以改善炎症损伤引起的内源性H2S合成的障碍,其中CBS可能是影响睾丸中内源性H2S水平主要的限速酶。小鼠睾丸行冰冻切片,DHE荧光染色测定超氧阴离子(02-)水平,结果显示,H2S可抑制睾丸02的生成。Tunel染色提示H2S可减轻炎症引起的生精细胞的凋亡。免疫荧光测定血睾屏障紧密连接蛋白,结果显示,H2S可维护血睾屏障的完整。ELISA方法检测小鼠血清睾酮水平,H2S能够抑制炎症造成的血清睾酮水平的降低。进一步研究H2S保护精子发生的的机制：Real-Time PCR的结果表明,H2S降低了炎症引起的睾丸中促炎因子IL-1p、IL-6、TNF-amRNA水平的升高,并提高抗炎因子ILˉ10的mRNA水平,同时改善炎症引起的睾酮合成相关蛋白StAR、P450scc、3β-HSD和P450c17mRNA水平的降低。试剂盒检测睾丸中的MDA水平,GSH/GSSG,以及SOD和caspase-3的活性,结果表明H2S可抑制炎症引起的MDA水平的增高,并维持GSH/GSSG的水平和SOD的活性,同时抑制caspase-3的激活。Western blot检测结果发现,H2S可以抑制IκB的降解和NF-κB的核转位(凝胶迁移率实验进一步证实H2S抑制了炎症状态下NF-κB活性的增高),抑制iNOS的表达,促进Nrf2的核转位并提高Nrf2在胞浆中的水平,H2S可以提高Bcl2/Bax的水平,同时H2S能够显著抑制MAPK信号通路蛋白JNK、ERK、P38的磷酸化水平。为了进一步证实内源性H2S对睾丸精子发生的意义,我们利用腺病毒转染的方式构建右侧睾丸组织过表达CBS的小鼠模型,在LPS刺激的条件下,CSAS的检测结果显示CBS过表达的右侧附睾中,精子质量明显高于左侧附睾。同时HE染色和免疫荧光染色的结果提示,在LPS造成炎症损伤的条件下,CBS过表达的右侧睾丸,其生精结构和血睾屏障的完整性与左侧睾丸相比均有明显改善。我们利用人精子细胞实验观测H2S对人类精子活力的影响：CASA检测结果显示内源性H2S合成酶CBS抑制剂AOAA显著抑制正常精子的活力水平和超激活运动能力,而外源性补充H2S能够改善CBS被抑制引起的精子活力和超激活运动能力的下降；合适浓度的H2S对于改善精浆中H2S水平较低的弱精症病人的精子活力具有一定效果。以上结果表明：①H2S对男性精子功能具有重要意义,外源性补充H2S有助于改善因H2S缺乏造成的精子功能障碍。②动物实验研究结果表明,H2S能够改善炎症和糖尿病引起的精子质量的下降,保护睾丸的生精结构,维持血睾屏障的完整,改善睾酮合成的障碍,维护正常精子发生。③H2S对精子发生和功能障碍的改善,依赖其可以通过抑制NF-κB的活性,控制睾丸组织中的炎症水平；通过促进Nrf2的核转位,提高抗氧化因子的表达抑制睾丸组织中氧化应激水平的增高；通过抑制线粒体诱导的凋亡信号通路的激活,减轻生精细胞的凋亡；通过抑制MAPK信号通路的激活,调控睾酮的合成和血睾屏障的完整。本研究为男性生育障碍的防治提供了新的思路。
[Abstract]:Male infertility is an important cause of human reproductive disorders. More than 85% of male sterile men can produce sperm, but most of them have low quality. The abnormal process of spermatogenesis affects sperm function and causes male infertility. In addition to genetic defects such as gene mutation and chromosome abnormality, environmental factors and infection factors are the causes of sperm. The main cause of occurrence and dysfunction. This is the most critical mechanism of oxidative stress and inflammation. So far, male infertility is clinically lacking in effective or targeted treatment. Hydrogen Sulfide (H2S) is third after nitric oxide (Nitic Oxide, NO) and carbon monoxide (Carbon Monoxide, CO). In mammals, in mammals, H2S mainly catalyzes the formation of L- cysteine by cystathion - gamma lyase (CSE) and cystathion -p synthetase (CBS). It is proved that the physiological concentration of H2S is of great physiological significance to scavenging oxygen free radicals, controlling the oxidative stress level of the body, and against the inflammatory reaction. In the study of female reproduction, H2S is of great significance to the development and maturation of egg cells in the study of female reproduction, and is of great value for the protection of fetal birth and pregnant women's cardiovascular function. In the male reproductive system, the H2S is the same. There are also endogenous H2S synthetases, but reports on their physiological significance are mostly limited to improving male erectile dysfunction. Endogenous H2S synthetases are expressed in male testicles and spermatozoa, and may have important potential value in regulating male spermatogenesis and sperm function. So far, H2S has been used for male birth. The main objective of this study is to explore the protective effect of H2S on the process of spermatogenesis and sperm function, and to further study the possible mechanism of its role. First, we used a free radical detector to evaluate H2S in normal human seminal plasma and fertility. The differences in the content of the seminal plasma in the lower patients were compared. The differences in the expression of endogenous H2S synthase in different sources were compared by Western-blot. The results showed that the seminal plasma of the low level H2S was often accompanied by poor vitality, and the expression of endogenous H2S synthase CBS in the weak spermatozoa decreased significantly, suggesting that H2S might be involved in male infertility. Pathological process. We use animal experiments to further verify the significance of H2S for male reproduction: we use LPS induced inflammation and STZ induced diabetes to establish a common spermatogenesis disorder model. After the modeling success, two different H2S donors are given intraperitoneally to GYY4137 or NaHS. After the drug is finished, computer assisted semen analysis is used. The quality of spermatozoa in the tail of epididymis of mice was evaluated by CASA, and the changes of spermatogenic structure in mice testis were observed by HE staining. The results showed that H2S could significantly inhibit the decrease of sperm quality caused by inflammation and diabetes and improve the damage of testicular structure. The endogenous H2S synthase CSE and CB were detected by Real-Time PCR and Western-blot method. The level of mRNA and protein expression of S and the catalytic activity of CBS and CSE in testis were detected by chemical method. The results showed that exogenous H2S could improve the obstacle of endogenous H2S synthesis caused by inflammatory injury. CBS may be the limiting enzyme that affects endogenous H2S level in the testis. Frozen section of mice testis and DHE fluorescence staining The level of superoxide anion (02-) was measured. The results showed that H2S could inhibit the formation of.Tunel in testis 02, suggesting that H2S could reduce the apoptosis of spermatogenic cells caused by inflammation. The immunofluorescence assay of blood testis barrier closely linked protein, the results showed that H2S could maintain the serum testosterone level of mice by the complete.ELISA method of maintaining the blood testis barrier, and H2S could inhibit the inflammation. The decrease in serum testosterone levels caused by the disease. Further study the mechanism of H2S protection of spermatogenesis: the results of Real-Time PCR suggest that H2S reduces inflammation induced IL-1p, IL-6, TNF-amRNA level, increases the mRNA level of IL - 10, and improves the testosterone synthesis associated protein StA of inflammation. R, P450scc, 3 beta -HSD and P450c17mRNA levels decreased. The kit detected the MDA levels, GSH/GSSG, and the activity of SOD and Caspase-3 in the testis. The results showed that H2S could inhibit the increase of MDA levels caused by inflammation, and maintain GSH/GSSG level and SOD activity. The degradation of I kappa B and the nuclear transposition of NF- kappa B (gel migration rate experiments further confirmed that H2S inhibited the increase of NF- kappa B activity in the inflammatory state), inhibited the expression of iNOS, promoted the nuclear transposition of Nrf2 and enhanced the level of Nrf2 in the cytoplasm, so that H2S could increase the level of Bcl2/Bax. In order to further confirm the significance of endogenous H2S to testicular spermatogenesis, we constructed the mouse model of the right testicular tissue over expression of CBS by adenovirus transfection. Under the condition of LPS stimulation, the results of CSAS detection showed that the quality of the right epididymis in the right epididymis was significantly higher than that of the left epididymis by CBS. Meanwhile, the sperm quality was significantly higher than that of the left epididymis. At the same time, HE staining was found. The results of color and immunofluorescence staining suggest that under the condition of LPS causing inflammatory damage, CBS overexpressed right testicles, the integrity of the spermatogenic structure and the blood testis barrier have improved significantly compared with the left testis. We used human sperm cells to observe the effect of H2S on human sperm motility: CASA results showed endogenous H2S The enzyme CBS inhibitor AOAA significantly inhibits the activity of normal spermatozoa and the activity of hyperactivation. Exogenous H2S can improve the decrease of sperm motility and hyperactivation induced by the inhibition of CBS, and the appropriate concentration of H2S can improve the sperm vitality of the asthenosin patients with low H2S level in the seminal plasma. The results showed that: (1) H2S is of great significance for male sperm function. Exogenous supplementation of H2S helps to improve sperm dysfunction caused by H2S deficiency. 2. Experimental results showed that H2S could improve the decrease of sperm quality caused by inflammation and diabetes, protect the spermatogenesis structure of the testis, maintain the integrity of the blood testis barrier and improve the integrity of the blood testis barrier. The obstacle of testosterone synthesis to maintain normal spermatogenesis. (3) the improvement of spermatogenesis and dysfunction by H2S depends on the inhibition of the activity of NF- kappa B to control the level of inflammation in the testicular tissue; by promoting the nuclear transposition of the Nrf2, the expression of antioxidant factors is increased to inhibit the increase of oxidative stress in the testis tissue; The activation of apoptosis signaling pathway induced by mitochondria reduces the apoptosis of spermatogenic cells, regulates the synthesis of testosterone and the integrity of the blood testis barrier by inhibiting the activation of MAPK signaling pathway. This study provides a new way of thinking for the prevention and control of male fertility disorders.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R698.2

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